In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.


Abstract:
CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize it's in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC 50 of CCL21 on MCF7 cells was less than that of nonrecombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.
Order  Yes -all data are fully available without restriction    Since the three dimensional (3D) structure of the recombinant protein is not available, comparative 124 modeling method was initially used to create the 3D structure of the recombinant protein.

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Comparative modeling is one of the best approaches to prediction of the 3D structure of the target 126 protein, in which 3D structures with sequences very similar to the target sequence are used as 127 templates. One of the best software for comparative modeling is MODELLER. The authors used 128 Modeller 9.24 for 3D modeling of the target . Ten models were produced, and the one with the 129 highest interaction and connectivity between the aforementioned epitopes was selected as the best 130 model [31]. performance. Finally, simulation data were stored at 0.4 pc intervals for analysis [28]. Two of the 158 best methods for such analyses are root mean square deviation (RMSD) and Radius of Gyration This project aimed to investigate the interaction between chemokine ligands and CCR7 receptors. 167 The CCR7 protein is intermembrane and, therefore, to simulate the CCR7-CCL21 complex, this 168 complex must first be located inside the plasma membrane. For this purpose, the CHARMM-GUI  Sterile leaf disks (about 0.5 cm 2 ) were excised and inoculated with recombinant Agrobacterium 199 tumefasciens [33]. Explants were transferred to a co-culture medium (MS with 0.2 mg/L BA and  x-y plotting, followed by fitting the corresponding data with a straight line (linear regression). The

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IC50 value was then estimated using the fitted line [39].  to the gel (1-6 hours before performing the cell migration assay) and three small wells with a 331 distance of 10 mm were cut in culture medium/ in each petri dish [40,43]. Germany) for 10 min [40]. The stained cells were then counted using a fluorescent microscope.

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Chromatin condensation and nuclear fragmentation were the criteria to confirm apoptosis. The final recombinant construct was codon-optimized. Codon Adaptation Index [44] was used for 350 assessing the degree of codon optimization of this construct and for increasing the protein 351 expression level; CAI 3 =1 was selected to support tobacco codon adaptation. A GC content of 48% 352 was accepted as an appropriate expression ( Figure 1A). Afterward, the synthetic ccl21/IL1β 353 construct was cloned into the binary vector pBI121 [45].   (Table 1). Epitopes with HLA coverage above 90% and IC50 374 below 50 were selected as the best epitopes for binding to MHCI and MHCII molecules. In

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A total of ten models were generated using Modeler 9.24, one of which was selected as the most  Figure 1C.

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The slope of RMSD increased rapidly in the first 10 ns of MD simulation. After approximately 385 10000 ps, RMSD reached 1 nm, which means that RMSD is equal to 1.25 nm at 60000 ps.  As mentioned, the purpose of this project was to investigate the interactions between ligands and 400 CCR7 receptors. As shown in Table 2, molecular docking was performed and the best cluster had a 401 score of -27, with a size of 34 complexes. In addition, Z-score was equal to -2.3. The image of this 402 complex is shown in Figure 1E. The RMSD parameter was used to study the stability of the 403 recombinant protein that bonded to the CCR7 receptor in molecular dynamics simulations and 404 using the RMSD parameter. The corresponding RMSD diagram and the final structure are shown in 405 Figures 1E and 1F. Figure 1E depicts the final structure of the MD complex, and it should be noted 406 that water and membrane molecules have been removed in the image to show the protein structure 407 more clearly. Figure 1F also represents the changes in the RMSD of the docked proteins over the The CCR7 protein is intermembrane and therefore, the CCR7-CCL21/IL1β complex must be  were observed for DNA samples of transgenic lines ( Figure 2B). Results indicated that the foreign gene was transcribed in the transgenic lines ( Figure 2C).  Figure 3B). In western blot analysis, the protein with an estimated 457 molecular weight of 55 kDa was successfully detected ( Figure 3C).

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Besides, ELISA assay was employed to quantify the expression of recombinant protein, and a 459 standard curve was drawn by ELISA using concentrations of commercial CCL21 (     was achieved using the Heit and Kubes method [47]. In this analysis, the number of migrated cells 549 in the distance of wells containing cells and those containing chemoattractants was deduced from 550 the number of cells that migrated between the wells containing cells and the wells containing non-551 absorbent material.Consequently, as shown in Figure 7, the number of monocytes moving toward 552 the adsorbent substance was higher than that toward the non-adsorbent material, and the chemotaxis 553 of the recombinant protein was estimated to be about 91% (Table 3). This modified methodology 554 allowed us to stain the nuclei of live cells by DAPI staining and use fluorescence microscopy to 555 overcome the issue of low contrast between the agar background and the cells. However, in our 556 experience, about 50% to 70% of the migrating cells might be lost due to cell staining ( Figures 7E   557 to 7I). Therefore, by examining the PBMC cells that had migrated toward the commercial CCL21, 558 the recombinant plant CCL21, and 10% FBS (as the growth factor), we found that CCL21 is a  In SDS PAGE, dot blotting and western blotting assays, the purified protein sample from transgenic 578 leaves generated a strong signal comparable to the positive control, whereas the protein from wild 579 type plant was not detectable.

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In previous studies on expression of other recombinant proteins in tobacco plants, TSP yields of 581 0.019-0.31% were reported [48,49]. In this work, however, CCL21/IL1β was expressed in the studying cellular chemotactic reactions and the signaling processes underlying them [54], [55]. 615 Generally speaking, chemokinesis is a chemically induced motile response of unicellular organisms 616 to chemical materials that proved the cells to alter their migratory behaviors. However, 617 chemokinesis has a a more random nature compared to chemotaxis [47]. Hence, the results of our 618 study demonstrate a differentiation between chemokinetic (CCL21) and chemotactic (FBS) 619 movement. It is expected that this recombinant multi-epitope protein can be considered for cancer 620 treatment. It can also treat viral diseases, such as AIDS and coronaviruses (e.g. Covid-19) since it 621 triggers the immune system to produce potent antibodies in patients with these diseases.